Real-time qPCR allows real time fluorescence detection and measurement of DNA amplification, and is often used for accurate quantitation of input DNA template in many molecular biology and biotech applications. There are several real-time qPCR chemistries and the most used two are: dsDNA binding (intercalating) fluorescence dyes, such as SYBR Green, and the 5′-nuclease hydrolysis fluorescence probe (TaqMan) method. The TaqMan method allows using multiple fluorescence report dyes, such as FAM, VIC, NED, 5- and/or 6-carboxy-X-rhodamine (e.g., available commercially as ROX™ and as SuperROX™ (from Biosearch Technologies, Petaluma, Calif.)), and CY5, each of which have different emission spectra for detection and measurement of multiple target DNA sequence amplifications in a single reaction.
There are many different real-time instrument platforms, such as 7500 and 7900 from Applied Biosystems, iQ5 and CFX96/384 from Bio-Rad, and some of them require an internal passive reference fluorescence dye for reporter dye fluorescence signal normalization from well to well to improve quantitation accuracy. Fluorescent dye ROX or a FRET ROX are often used as passive reference dyes (see, e.g., U.S. Pat. No. 5,736,333) in qPCR and RT-qPCR master mix products. Due to differences in design of thermal cyclers, some instruments employ a high concentration of 5- or 6-carboxy-X-rhodamine dye for normalization while others employ low concentration. The normal ROX dye has an excitation/emission maximum at 586/610 nm and can be excited efficiently on the 7500 and other “low ROX” instruments, which provide different excitation wavelength lights for each different fluorophore channels. The ROX dye cannot be excited efficiently on the 7900 “high ROX” instrument, which provides only a single 488 nm laser light for excitation of all different fluorophores, and hence a higher concentration of ROX is needed to generate required reference signal intensity for normalization. Typically, the ROX concentration used by a “high-ROX” platform is about 10-fold higher than that used by a “low-ROX” platform.
A FRET ROX is a FAM/ROX FRET-based oligo conjugation that can be excited both at 586 nm maximum as normal ROX dye, and at around 488 nm for the FAM fluorophore that upon being excited transfers the energy to the ROX fluorophore to emit fluorescence at 610 nm maximum. See, e.g., U.S. Pat. No. 5,736,333. A qPCR master mix with a single concentration of FRET ROX can be used on all different ROX-dependent instruments, whether it is a low ROX or high ROX instrument. A qPCR master mix with a defined concentration ratio of a normal ROX dye and a long Stokes-shift fluorescence dye that has an emission spectrum maximum similar to ROX but can be excited at around 488 nm can serve as a universal ROX reference dye similar to the FRET ROX, and can be used on different ROX-dependent qPCR instruments. See, e.g., US Patent Publication No. 2012/0164690.
PCR and other nucleic acid amplification reaction set-up on microwell-plate can involve pipetting a (1) reaction master mix that contains all assay components except the nucleic acid template, and (2) a DNA sample that contains the nucleic acid (e.g., DNA) template component separately into reaction wells. When this is done on a microwell plate with small volumes, it is easy to lose track of whether or not a component has been pipetted into a well, especially when a white or non-transparent well plate is used. Thus, reaction set-up errors can occur, resulting in failed PCR reactions. PCR master mixes with inert visible dye have been used to minimize pipetting errors for routine PCR and real-time PCR, and commercial products are available, such as RedTaq ReadyMix PCR Reaction Mix of Sigma Aldrich, Absolute Blue qPCR Mixes, DyNamo ColorFlash qPCR Kits, and Luminaris Color qPCR Master Mixes of ThermoScientific, and TaqMan GTXpress Master Mix (with tracking dye) of Life Technologies.